Cancer antigen-specific t-cell receptor gene, peptide encoded by the gene, and use of them

ABSTRACT

Disclosed are: a nucleotide sequence and an amino acid sequence for CDR3 region of T-cell receptor (TCR) gene of WT1-specific cytotoxic T-cell (CTL) for WT1 protein; a method for the detection or treatment of cancer using the nucleotide sequence or the amino acid sequence; and a chip, a primer set, a kit, an apparatus and the like for use in the detection of cancer, each of which comprises the nucleotide sequence or the amino acid sequence.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional application of U.S. patent application Ser. No.14/184,979, filed Feb. 20, 2014, which is a divisional application ofU.S. application Ser. No. 12/529,701, which issued on Oct. 9, 2018, asU.S. Pat. No. 10,093,977, whose 35 U.S.C. § 371(c) date is Mar. 26, 2010and which is a national stage of International Application No.PCT/JP2008/053469, filed Feb. 28, 2008, all of which claim priority toJapanese Patent Application No. 2007-054215, filed Mar. 5, 2007, and theentire disclosures of all of which are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format in U.S. patent application Ser.No. 14/184,979 and is hereby incorporated by reference in its entirety.Said ASCII copy, created on Feb. 14, 2017, is named 05273 0120-02000SL.tzt and is 582,545 bytes in size.

TECHNICAL FIELD

The present invention relates to a polynucleotide contained in the genefor a cancer antigen-specific T-cell receptor, and the peptide encodedby the polynucleotide, and to cancer tests, e.g., diagnosis, prognosis,or treatment monitoring using the same, cancer therapy, and the like.

BACKGROUND ART

Cancer treatments include extirpation of cancer by surgical operation,radiotherapy, treatment with anti-cancer medication, and immunotherapy.Among these treatments, immunotherapy in particular has drawn attentionin recent years because it is a treatment more selective and specificagainst cancer with least side effects. Inter alia, attempts have beenextensively made to treat cancer and leukemia by targeting WT1 protein,which is abundantly present in many types of cancer cells and leukemiacells. In order to study the mechanism of the WT1-targeted anti-cancertherapy and to further increase the effect of the therapy, it isnecessary to identify the nucleotide sequences of the T-cell receptor(TCR) genes of WT1-specific cytotoxic T-cells (CTL) and the amino acidsequences of the receptor peptides encoded by the nucleotide sequences.However, up to the present date, little is known about the sequences ofthose receptor genes and the receptor peptides, let alone that noeffective use of them has been described, although a number ofresearches have been conducted (see, e.g., non-patent documents 1-5).

Non-Patent Document 1: Valmori D. et al., J. Immunol. 168: 4231-4240,2002

Non-Patent Document 2: Dietrich P . et al., Cancer Res. 61: 2047-2054,2001

Non-Patent Document 3: Coulie P . et al., Proc. Natl. Acad. Sci. U.S.A.98: 10290-10295, 2001

Non-Patent Document 4: Godelaine D. et al. J. Immunol. 171: 4893-4897,2003

Non-Patent Document 5: Mandruzzato S. et al., J. Immunol. 169:4017-4024, 2002

SUMMARY OF THE INVENTION

An object of the present invention was to reveal the amino acidsequences of T-cell receptors (TCRs) of WT1-specific cytotoxic T-cells(CTLs) for WT1 protein, and the nucleotide sequences of the genesencoding them, in particular, the amino acid and nucleotide sequences ofthe CDR3 region of them, as well as to use those pieces of informationin cancer tests (diagnosis, prognosis, treatment monitoring, or thelike) and in cancer therapy.

DISCLOSURE OF INVENTION Problems to be Solved by the Invention

To accomplish the above object, the present inventor has conductedextensive research and determined, for the first time, the nucleotideand amino acid sequences of CD3 of the Vβ chain of T-cell receptors(TCRs) of WT1-specific cytotoxic T-cells (CTLs) from healthy individualsand HLA-A*2402-positive patients, and have thereby completed the presentinvention.

Thus, the present invention provides the following:

-   (1) A polynucleotide having a nucleotide sequence encoding CDR3 of a    Vβ chain of a T-cell receptor (TCR) of a WT1-specific cytotoxic    T-cell (CTL), said polynucleotide having DNA having any of the CDR3    nucleotide sequences shown in SEQ ID Nos.: 1-1696, RNA complementary    to the DNA, or a polynucleotide having a complementary sequence    thereof;-   (2) The polynucleotide according to (1), wherein said polynucleotide    consists of a DNA comprising any of the CDR3 nucleotide sequences    shown in SEQ ID Nos.: 1-1696, or an RNA complementary thereto, or    complementary sequences thereof;-   (3) A peptide having an amino acid sequence of CDR3 of a Vβ chain of    a T-cell receptor (TCR) of a WT1-specific cytotoxic T-cell (CTL),    said peptide having any of the amino acid sequences of CDR3 shown in    SEQ ID Nos.: 1697-3392;-   (4) The peptide according to (3) consisting of any of the amino acid    sequences of CDR3 shown in SEQ ID Nos.: 1697-3392;-   (5) Use of the polynucleotide of (1) or (2), or the peptide of (3)    or (4) as a cancer marker;-   (6) A method for diagnosing cancer in an HLA-A*2402-positive    patient, which comprises assessing the clonality of a WT1-specific    CTL having any of the polynucleotides of (1) or (2), or any of the    peptides of (3) or (4) in a sample obtained from the patient before    therapy, wherein when a WT1-specific CTL having a multiplicity of    clonality is present, the patient is determined to have a    possibility of having cancer;-   (7) The method according to (6), wherein a higher possibility of    developing cancer in the patient before therapy is determined when    the clonality of a WT1-specific CTL having any of the CDR3    polynucleotides or any of the CDR3 peptides and having the clonality    of 3 or more is higher, or when the types of a WT1-specific CTL    having the clonality of 3 or more are more abundant;-   (8) A method for testing for sensitivity of an HLA-A*2402-positive    patient to WT1 peptide immunotherapy, which comprises assessing the    clonality and the number of types of WT1-specific CTLs having any of    the polynucleotides of (1) or (2) or any of the peptides of (3)    or (4) in a sample obtained from the patient before therapy, wherein    the patient is determined to have sensitivity to WT1 peptide    immunotherapy when the types of WT1-specific CTLs with a    multiplicity of clonality are more abundant in the patient than in a    subject non-responsive to the therapy;-   (9) The method according to (8), wherein a patient is determined to    have higher sensitivity to WT1 peptide immunotherapy when the types    of WT1-specific CTL clones with a multiplicity of clonality are more    abundant, or the clonality is larger in the patient before the    therapy;-   (10) A method for monitoring WT1 peptide immunotherapy in an    HLA-A*2402-positive patient, which comprises assessing the clonality    of WT1-specific CTL clones having any of the polynucleotides of (1)    or (2) or any of the peptides of (3) or (4) in a sample obtained    from the patient before and after the therapy, wherein the patient    is determined to have responded to WT1 peptide immunotherapy when    the clonality of any of the WT1-specific CTL clones increases after    the therapy compared to before the therapy;-   (11) The method according to (10), wherein a patient is determined    to have higher responsiveness to WT1 peptide immunotherapy when the    larger becomes the increase rate in the clonality, or the more    abundant become the types of clones with increased clonality after    the WT1 peptide immunotherapy;-   (12) An antibody against the peptide of (3) or (4);-   (13) A chip comprising the polynucleotide of (1) or (2), the peptide    of (3) or (4), or the antibody of (12);-   (14) A primer for amplifying a CDR3 polynucleotide, which has a    sequence selected from the sequences shown in SEQ ID Nos.:    3394-3421;-   (15) A kit for diagnosing cancer, a kit for testing for sensitivity    of a patient to WT1 peptide immunotherapy, or a kit for monitoring    WT1 peptide immunotherapy, comprising means for detecting a    WT1-specific CTL having the polynucleotide of (1) or (2) or the    peptide of (3) or (4);-   (16) A device for cancer diagnosis, a device for testing for    sensitivity of a patient to WT1 peptide immunotherapy, or a device    for monitoring WT1 peptide immunotherapy, comprising means for    detecting a WT1-specific CTL having the polynucleotide of (1) or (2)    or the peptide of (3) or (4);-   (17) The kit according to (15), comprising the chip of (13) or the    primer of (14);-   (18) The device according to (16), wherein the chip of (13) or the    primer of (14) is used in the device;-   (19) A lymphocyte of an HLA-A*2402-positive cancer patient, into    which a T-cell receptor gene comprising a sequence of the    polynucleotide of (1) or (2) is introduced.

The present invention also provides cancer therapy using lymphocytesfrom HLA-A*2402-positive patients, wherein a T-cell receptor genecontaining a CDR3 polynucleotide is introduced into the lymphocytes.

Further, the present invention provides an antibody raised against aCDR3 polypeptide and use thereof.

Effect of the Invention

By virtue of the present invention, the nucleotide sequences containedin the gene for the Vβ chain of T-cell receptors (TCRs) of WT1-specificcytotoxic T-cells (CTLs), and the amino acid sequences of peptidesencoded by them, in particular, the nucleotide and amino acid sequencesof CDR3 have been revealed, and extensive cancer tests (diagnosis,prognosis, treatment monitoring, or the like), cancer therapy, and thelike are enabled using these polynucleotides and peptides.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1 through 1ZZZ-3 comprise a table that shows the nucleotide andamino acid sequences of CDR3 of the Vβ chain of T-cell receptors (TCRs)of WT1-specific cytotoxic T-cells (CTLs) from healthy individuals andHLA-A*2402-positive patients, which sequences have been identified inthe present invention. In the figures, “clone #” indicates the clonenumbers, the numbers to the left of the dot indicate the family numbersof Vβ chains, and the numbers to the right of the dot indicate thereference numbers. V name, J name, and D name indicate the details ofthe V region, J region, and D region, respectively. The letters“V-REGION...N1...D-REGION...(P)N2...J-REGION” on the nucleotide sequenceindicate the origins of each portion of the nucleotide sequence.

In FIGS. 1A-1 through 1ZZZ-3, the nucleotide sequences shown under“V-REGION...NI...D-REGION...(P)N2...J-REGION” correspond to SEQ ID NOs:1-1696 as identified in the Sequence Listing, and the amino acidsequences shown under “CDR3 amino acid” correspond to SEQ ID NOs:1697-3392 as identified in the Sequence Listing.

FIGS. 2A-1 through 2VVVV-3 comprise a table that shows the clonality ofeach clone of the WT1-specific cytotoxic T-cells (CTLs) from healthyindividuals and HLA-A*2402-positive patients identified in the presentinvention. In the figure, “clone #” indicates the clone numbers, thenumbers to the left of the dot indicate the family numbers of Vβ chains,and the numbers to the right of the dot indicate the reference numbers;HV is healthy volunteers, AML is patients with acute myelocyticleukemia, and MDS is patients with dysmyelopoietic syndrome. The numbersadded to these abbreviations are healthy volunteer numbers and patientnumbers. HV is healthy volunteers, AML is patients with acute myelocyticleukemia, and MDS is patients with dysmyelopoietic syndrome. The numbersadded to these abbreviations are healthy volunteer numbers and patientnumbers. Res indicates patients who responded to treatment; and nonResindicates patients who did not respond to treatment. Further, preindicates pre-treatment, and 4 w, 8 w, 12 w, and 42 w indicate 4 weeks,8 weeks, 12 weeks, and 42 weeks, respectively, after the treatment.Phase 1 denotes the first phase of clinical trials. PB(pre) indicatesperipheral blood before treatment; and BM(pre) indicates bone marrowbefore treatment.

BEST MODE FOR CARRYING OUT THE INVENTION

The present inventor stained the peripheral lymphocytes from cancerpatients with WT1 tetramer that comprises WT1 peptide/HLA-A*2402complexes, and separated WT1 tetramer-positive cells one by one using aFACS; cDNAs were generated from each separated cell, and the nucleotidesequences encoding CDR3 contained in the Vβ chain of T-cell receptors(hereinafter may be referred to as “TCR”) of WT1-specific cytotoxicT-cells (CTLs, hereinafter may be referred to as “WT1-sepcific CTL”)were determined by applying PCR method (FIGS. 1A-1 to 1ZZZ-3). Fromthese results, the amino acid sequences of the CDR3 were also determined(FIGS. 1A-1 to 1ZZZ-3; SEQ ID Nos.: 1697-3392). These sequences havebeen determined for the first time in the present invention.

Thus, in a first aspect, the present invention provides a polynucleotidehaving the nucleotide sequence encoding CDR3 of the Vβ chain of T-cellreceptors (TCRs) of WT1-specific cytotoxic T-cells (CTLs) obtained fromhealthy individuals and HLA-A*2402-positive patients, wherein thepolynucleotide is DNA having any of the CDR3 nucleotide sequences shownin SEQ ID Nos.: 1-1696, RNA complementary to the DNA, or apolynucleotide having a complementary sequence thereof. As used herein,these DNA and RNA molecules and the complementary polynucleotidesthereof are collectively referred to as “CDR3 polynucleotides.” Forexample, the CDR3 polynucleotides include, in addition to the DNAscomprising the nucleotide sequences shown in SEQ ID Nos.: 1-1696, DNAscomprising these sequences. Also, for example, the CDR3 polynucleotidesinclude, in addition to the RNAs complementary to the DNAs comprisingthe nucleotide sequences shown in SEQ ID Nos.: 1-1696, RNAs comprisingthese sequences. Further, for example, the CDR3 polynucleotides includepolynucleotides having the sequences of the DNAs or RNAs, andpolynucleotides complementary to the RNAs. The “CDR polynucleotides”include those having degenerate sequences encoding “CDR3 peptides.”

In another aspect, the present invention provides peptides having theamino acid sequences of CDR3 of the Vβ chain of T-cell receptors (TCRs)of WT1-specific cytotoxic T-cells (CTLs), which CDR3 amino acidsequences being shown in any of SEQ ID Nos.: 1697-3392. As used herein,these peptides are collectively referred to as “CDR3 peptides.” Forexample, the CDR3 peptides include, in addition to a peptide comprisingany of the CDR3 amino acid sequences shown in SEQ ID Nos.: 1697-3392,peptides comprising these CDR3 amino acid sequences (such as, forexample, Vβ chain peptides or a portion thereof). In addition, a peptideconsisting or comprising the amino acid sequence shown in SEQ ID Nos.:1697-3392 in which one or a few, preferably one to three, one or two, orone amino acid is substituted, added, or deleted is included in the“CDR3 peptides.” However, these peptides are required to have equivalentfunctions to the original peptide. These CDR3 peptides are encoded bythe above-mentioned CDR3 polynucleotides.

The CDR3 regions are the most diverse portions and are the mostresponsible parts for the specificity of antigen recognition. Thus, thesequences of the CDR3 polynucleotides and CDR3 peptides of the presentinvention are considered peculiar to WT1-specific CTL inHLA-A*2402-positive patients. Therefore, provided that thepolynucleotide encoding the CDR3 region of the gene for the TCR Vβ chainof a certain T-cell or the peptide corresponding to the CDR3 region havethe sequence of the polynucleotide or peptide of the present invention,the T-cell is considered as specific for WT1. Accordingly, the CDR3polynucleotides and CDR3 peptides of the present invention may find useas markers for a wide variety of cancers, use in applications such asdiagnosis of cancer, diagnosis of the susceptibility of patients to WT1peptide immunotherapy, and tests for the responsiveness in the patientsto WT1 peptide immunotherapy.

The CDR3 polynucleotides and CDR3 peptides of the present invention maybe present in the lymphocytes of patients with any type of cancer aslong as the cancer is generated from cells containing WT1. WT1 is knownas a cancer antigen in a variety of cancers and hematologicalmalignancies. Thus, the CDR3 polynucleotides and CDR3 peptides of thepresent invention, as well as the methods of the present inventiondescribed below, may be applied to almost all the types of cancersincluding, but not limited to, for example, hematologic malignancies,such as acute myelocytic leukemia, acute lymphocytic leukemia, malignantlymphoma, multiple myeloma, chronic myelocytic leukemia, myelodysplasticsyndrome, and recurrence after the transplantation of hematopoietic stemcells of the same type; solid cancers, such as tongue cancer, gingivalcancer, mouth floor cancer, pharyngeal cancer, larynx cancer, salivarygland cancer, and thyroid cancer; thoracic cancers, such as breastcancer, lung cancer, and thymic cancer; gastrointestinal cancers, suchas colon cancer, small intestine cancer, gastric cancer, pancreaticcancer, liver cancer, bile duct cancer, gastrointestinal endocrinetumor, and gastrointestinal carcinoid; cancers of urinary and genitaltract, such as renal cancer, urothelial cancer, germinoma, Wilms' tumor,prostate cancer, uterine body cancer, cervical cancer, uterine sarcoma,and ovarian malignancy; musculoskeletal malignancies, such as primarymalignancy of bone (e.g., osteosarcoma and Ewing's sarcoma) and softtissue sarcoma; and other cancers, such as skin cancer, neuroblastoma,malignant glioma (glioblastoma), primary malignant lymphoma of thecentral nervous system, medulloblastoma, and PNET.

When producing CDR3 polynucleotides or CDR3 peptides, conventionalgenetic engineering techniques and/or chemical synthetic procedures maybe used. For example, CDR3 polynucleotides may be isolated from cells orchemically synthesized. CDR3 polynucleotides may also be amplified usinga known method, such as PCR. Also, for example, a CDR3 polynucleotide(optionally amplified to an appropriate level using a known method) maybe integrated into a suitable vector and introduced into suitable cells,or may be introduced into suitable cells by biolistic bombardment orelectroporation. Then the cells into which the CDR3 polynucleotide isintroduced are cultured for expression, thereby obtaining the CDR3polynucleotide or peptide. Available vectors and cells, conditions forgene transfer, culture conditions, and methods for isolating genes andpeptides are known to those skilled in the art and appropriatelyselected for use. Chemical synthesis may be used to produce CDR3polynucleotides or CDR3 peptides. Methods for such chemical synthesisare known, and the methods for chemical synthesis of genes includesolid-phase DNA synthesis using amidite, and the 1-4 phosphonate method;the methods for chemical synthesis of peptides include the Fmoc method.

Thus, in a further aspect, the present invention provides a method fordiagnosing cancer in an HLA-A*2402-positive patient, the methodincluding assessing the clonality of WT1-specific CTL having any of theCDR3 polynucleotides or CDR3 peptides in a sample obtained from thepatient before therapy, wherein if a multiplicity of clonality arepresent, the patient is determined to have a possibility of cancer. Asused herein, the term “clonality” refers to the frequency of detectionof cells having an identical nucleotide or amino acid sequence. Toexamine clonality, it is general to use a cell sorter that allowsidentification of individual cells. The “patients” include both humanssuspected to have cancer and those suffering from cancer.

The present inventor has found that it is possible to determine whethera patient develops cancer or not by examining the clonality ofWT1-specific CTL having any of the CDR3 polynucleotides or CDR3 peptidesin the patient. As can be seen from FIGS. 2A-1 to 2VVVV-3, in thehealthy individuals (HV1 to HV4), the clonality of WT1-specific CTLhaving any of the CDR3 polynucleotides or CDR3 peptides is 1 for almostall the clones, and 2 or 3 for rare clones; however, by contrast, allthe cancer patients before therapy (AMLs (acute myelocytic leukemia) 1-4and MDS (myelodysplastic syndrome) 1-5) have a multiplicity of clonalityof the WT1-specific CTLs having any of the CDR3 polynucleotides or CDR3peptides without exception. The number of the clonality is larger andthe types of such cells are more abundant than those of the healthyindividuals. The increases in clonality and in types of cells having amultiplicity of clonality in patients before therapy indicate apossibility that defense and attack against cancer cells has alreadybeen launched in the patients.

In view of these results, the possibility of cancer in a patient may bedetermined if a multiplicity of clonality is found in the WT1-specificCTL having any of the CDR3 polynucleotides or CDR3 peptides whenexamining a sample from a subject. Further, it is possible to determinethat the larger the clonality of WT1-specific CTL having any of the CDR3polynucleotides or CDR3 peptides, or the more abundant the types ofWT1-specific CTLs having a multiplicity of clonality, the higher thepossibility of developing cancer is in the subject before therapy. Also,in the determination method, it is possible to determine that the higherthe possibility of developing cancer in the patient before therapy iswhen the clonality of WT1-specific CTL having any of the CDR3polynucleotides or CDR3 peptides and having the clonality of 3 or moreis larger, or the types of WT1-specific CTL having the clonality of 3 ormore are more abundant.

In a further aspect, the present invention provides a method for testingfor the sensitivity of an HLA-A*2402-positive patient to WT1 peptideimmunotherapy, the method including assessing the clonality and thenumber of types of WT1-specific CTL clones having any of thepolynucleotides of claim 1 or any of the peptides of claim 2 in a sampleobtained from the patient before therapy, wherein the patient isdetermined to have the sensitivity to WT1 peptide immunotherapy when thetypes of WT1-specific CTL clones with a multiplicity of clonality aremore abundant in the patient than in non-responsive subjects.

WT1-specific CTLs having any of the CDR3 polynucleotides or CDR3peptides act against cancer cells in the patients. In this process, theclones that already have a multiplicity of clonality before the therapytend to further increase the clonality or maintain their clonality byWT1 peptide immunotherapy. It is also considered that WT1 immunotherapyis more likely to be successful when there are clones of as many typesas possible, which gives increased number and types of effectiveWT1-specific CTL clones as a whole. More strictly speaking, thesensitivity of a patient to WT1 peptide immunotherapy may be determinedas being high when the types of WT1-specific CTL clones with amultiplicity of clonality are more abundant in the patient than innon-responsive subjects.

As can be seen from FIGS. 2A-1 to 2VVVV-3, the AML patients whoresponded to WT1 peptide immunotherapy had a larger number of the typesof clones with a multiplicity of clonality than the healthy subjects andnon-responsive patients before the therapy. AML1 (responder): 10 types(clonality of 58 in total); AML2 (responder): 12 types (clonality of 93in total); AML3 (non-responder): 9 types (clonality of 27 in total); andAML4 (non-responder): 3 types (clonality of 10 in total). Healthyindividuals HV1-5 had clones with a multiplicity of clonality in a rangeof 0 to 2 types.

In a further aspect, the present invention provides a method formonitoring WT1 peptide immunotherapy in an HLA-A*2402-positive patient,the method including assessing the clonality of WT1-specific CTL cloneshaving any of the polynucleotides of claim 1 or any of the peptides ofclaim 2 in a sample obtained from the patient before and after thetherapy, wherein the patient is determined to have responded to WT1peptide immunotherapy when the clonality of any of the WT1-specific CTLclones increases after the therapy compared to before the therapy.

As can be seen from FIGS. 2A-1 to 2VVVV-3, WT1-specific CTLs areobserved in which the clonality increased (including the cases where theclonality increased from 0 to 1 or more) in the AML patients during theperiod of WT1 peptide immunotherapy. The exemplary clones exhibitingsuch behavior include Clones #02.28, #05.034, #05.135, #05.219, #09.65,#12.04, #13.13, #27.016, and #28.83 from AML (Pt1), and Clones #02.27,#05.035, #05.141, #05.219, #06.009, #12.04, #12.20, #15.44, #20.068, and#27.032 from AML (Pt2). The presence of clones that increases theirclonality due to WT1 peptide immunotherapy indicates the responsivenessto the WT1 peptide immunotherapy. In other words, increases in theclonality suggests that the WT1 peptide immunotherapy showed its effectfor a certain period of time. These increases in clonality may betransient or sustained. If the clonality of certain clones may increaseonly temporarily and then decrease, the clonality of other WT1-specificCTLs would increase to complement the effect. Considering the effect oncancer cells, a larger increase in the clonality is more desired.Therefore, the larger becomes the increase in the clonality or the moreabundant become the types of clones with increased clonality after theWT1 peptide immunotherapy, the patient may be determined to have higherresponsiveness to the WT1 peptide immunotherapy. The number andproperties of cancer cells may be examined using an appropriate methoddepending on the type and site of the cancer.

In the method for monitoring therapy, although the clonality is comparedbefore and after WT1 peptide immunotherapy, the time period between thebefore and after the therapy may be of any length. For example, it maybe a few days, one week, two, three, or four weeks, or two or threemonths or more.

In the methods of the present invention described above, the means andmethods for assessing clonality and determining the types of clones(i.e., determining the amino acid sequences of CDR3 peptides or thenucleotide sequences encoding the same) are known in the art, and thoseskilled in the art may conveniently carry out these operations. Forexample, as shown in Examples in the specification, a known sortingapparatus, such as the FACSAria system, and a method for geneamplification, such as PCR (using, for example, a primer set selectedfrom the sequences listed in Table 1), may be used. In order to analyzethe CDR3 polynucleotides or CDR peptides of the present invention in astricter or more definite manner, one has only to confirm whether thenucleotide sequences of CDR3, IMGT, and the J and D regions in the Vβchain gene are shown in FIGS. 1A-1 to 1ZZZ-3. Such confirmation is wellwithin the ordinary skill of the art.

WT1 peptide immunotherapy is also known. For example, it may beperformed on HLA-A*2402-positive patients by administeringHLA-A*2402-restricted WT1 peptide in the native form (having the aminoacid sequence: CMTWNQMNL) or in the modified form (having the amino acidsequence: CYTWNQMNL) via, for example, a transdermal route. In general,a single dose is in the order of μg/kg body weight to mg/kg body weight,and it may be administered at an interval of one week to a few weeks.

In a further aspect, the present invention provides a chip comprisingthe CDR3 polynucleotides or the polynucleotides complementary thereto; achip comprising the CDR3 peptides; or a chip comprising antibodiesagainst the CDR3 peptides. The chips may be in the form of microchips,microarrays, and the like. The production of the chips may be conductedaccording to conventional methods; for example, antibodies raisedagainst the CDR3 polynucleotides or CDR3 peptides may be immobilized ona glass substrate. The species of the CDR3 polynucleotides or thepolynucleotides complementary thereto, the CDR3 peptides, or theantibodies against the CDR3 peptides that is immobilized on the chip maybe one to all; preferably, all the species are immobilized forexhaustive analysis. For example, all the polynucleotides comprising thenucleotide sequences complementary to the nucleotide sequences shown inSEQ ID Nos.: 1-1696 may be immobilized on the chip; alternatively, forexample, antibodies that specifically recognize and bind to all thepeptides comprising the amino acid sequences shown in SEQ ID Nos.:1697-3392 may be immobilized on the chip. The CDR3 polynucleotides orthe polynucleotides complementary thereto, the CDR3 peptides, or theantibodies against the CDR3 peptides may be placed at any position onthe chip.

The chips may be used for, for example, diagnosis of cancer as describedabove. The samples may be affected tissues, body fluids such as bloodand lymphatic fluid, or mucosal membranes. Preferably, the samples areperipheral blood. For example, when CDR3 polynucleotides are to beanalyzed, the nucleic acids are extracted from the cells according toconventional methods, and a chip onto which all the species ofpolynucleotides comprising the nucleotide sequences complementary to thenucleotide sequences shown in SEQ ID Nos.: 1-1696 are immobilized may beused to examine the species and quantity of the hybridized DNA presentin the sample. Also, for example, when CDR3 peptides are to be analyzed,a chip onto which antibodies that specifically recognize and bind to allthe species of peptides comprising the amino acid sequences shown in SEQID Nos.: 1697-3392 are immobilized may be used to examine the speciesand quantity of the specifically bound peptides present in the sample.

In this regard, the present invention provides an antibody thatspecifically recognizes and binds to a CDR3 peptide. Preferably, such anantibody specifically recognizes and binds to any of the amino acidsequences shown in SEQ ID Nos.: 1697-3392. Methods for preparing such anantibody are known to those skilled in the art.

Typically, DNAs in the sample or DNA sequences placed on the chip arelabeled so that the presence or absence, or the amount of hybridizationis indicated. For example, the presence or absence, or the species ofCDR3 peptides in a sample may be identified by arraying antibodies foreach of the CDR3 peptides of SEQ ID Nos.: 1697-3392 on a chip andtesting for their specific binding to the CDR3 peptides present in thesample. Typically, the peptides in the sample or the antibodies on thechip are labeled so that the presence or absence of the specific bindingcan be determined. Labels capable of indicating the presence or absenceand the amount of hybridization or specific binding are known andinclude, for example, fluorescent labels, radioactive labels, enzymelabels, and chromophore labels. One skilled in the art may convenientlyselect suitable labels. The chips described above may be used to analyzea plurality of samples at the same time.

The CDR3 polynucleotides and CDR peptides of the present invention maybe analyzed and identified using a known method, such as Southernblotting, Northern blotting, colony hybridization, and ELISA, as well asusing the chips described above.

As described above, the CDR3 DNAs of the present invention have beenidentified using the primers shown in Examples, particularly, the primersets shown in Table 1. Therefore, the present invention provides primersfor amplifying CDR polynucleotides, which primers having the sequencesselected from the sequences shown in SEQ ID Nos.: 3394-3421. Forexample, a primer set comprising the primers shown in SEQ ID Nos.:3396-3420 may be used for amplification of a CDR3 polynucleotide.

The present invention also provides a kit for diagnosing cancerincluding means for detecting a WT1-specific CTL having a CDR3polynucleotide or CDR3 peptide; a kit for testing for the sensitivity ofa patient to WT1 peptide immunotherapy; or a kit for monitoring WT1peptide immunotherapy. The present invention further provides a devicefor cancer diagnosis including means for detecting a WT1-specific CTLhaving a CDR3 polynucleotide or CDR3 peptide; a device for testing forthe sensitivity of a cancer patient to WT1 peptide immunotherapy; or adevice for monitoring WT1 peptide immunotherapy. A part for amplifyinggenes, such as a primer set, as described above, a chip as describedabove, or means for analyzing the information obtained from the chip maybe used in the kit as a component or in the device.

In still another aspect, the present invention relates to a lymphocytefrom an HLA-A*2402-positive cancer patient, which lymphocyteincorporating a T-cell receptor gene containing a sequence of a CDR3polynucleotide. Preferably, such a lymphocyte is a peripheral bloodlymphocyte into which the gene for the Vβ chain of TCR of WT1-specificCTLs comprising a CDR3 polynucleotide of the present invention, and agene for the Vα chain of TCR of WT1-specific CTLs. In preparation ofsuch a peripheral blood lymphocyte, a single species of the gene for theVβ chain of TCR of WT1-specific CTLs may be used to obtain a pluralityof types of peripheral blood lymphocytes, which are in turn introducedinto patients. However, in view of improving the therapeutic effect, itis preferred to use a plurality of species of the gene for the Vβ chainof TCR of WT1-specific CTLs to obtain a plurality of types of peripheralblood lymphocytes, which are in turn introduced into patients.Alternatively, it is also preferred to select the nucleotide sequencesof a suitable gene to be introduced depending on individualcircumstances, because the therapeutically effective nucleotidesequences in the gene may differ depending on patients and cancer types.

Methods for preparing a gene to be introduced and for introducing thegene into peripheral blood lymphocytes are known in the art. Forexample, a gene to be introduced may be integrated into a suitablevector and then introduced into suitable cells, or may be introducedinto suitable cells by biolistic bombardment or electroporation. Otherconditions for gene transfer and for cell culture may be appropriatelyselected by those skilled in the art.

The lymphocytes into which a gene has been introduced as described abovemay be cultured ex vivo to obtain a large amount of WT1-specific CTLs.Then, the CTLs obtained may be introduced into a cancer patient to killtumor cells expressing WT1, thereby performing cancer therapy. Patientspreferred for introduction of the CTLs obtained are the patients fromwhom the peripheral blood lymphocytes were obtained.

The cancer therapy described above may be combined with other cancertherapies including anti-cancer agents and radiotherapy. The cancertherapy described above has a wide range of applications. They areexemplified above, but are not limited thereto.

In still another aspect, the present invention provides an antibodyagainst a CDR3 peptide and a method of use thereof. Methods forpreparing such an antibody are known in the art. Such an antibody may beused to detect or identify a lymphocyte having the amino acid sequenceof the CDR3 peptide of the present invention or an amino acid sequencecontaining the above sequence in its Vβ chain in the subject sample. Forexample, antibodies against peptides comprising the amino acid sequencesof any of SEQ ID Nos.: 1697-3392 may be used to detect or identifycancer-specific lymphocytes. These antibodies may also be used to carryout the methods of the present invention, for example, the method fordiagnosing cancer.

Such an antibody may also be contacted with lymphocytes having the aminoacid sequence of CDR3 of the present invention to activate them. Thelymphocytes thus activated may be used to treat cancer. Preferably,lymphocytes obtained from a cancer patient are activated and, ifnecessary, proliferated, and the cancer therapy is conducted byreturning the lymphocytes to the patient. Such an antibody may also beused to enrich the WT1-specific T-cells of interest. For example, suchan antibody may be used to enrich the WT1-specific T-cells in a cancerpatient, thereby assisting the cancer therapy.

In a further aspect, the present invention provides a method foridentifying the position and size of a solid cancer, the methodincluding: administering the peripheral blood lymphocytes of the presentinvention described above after being labeled with a detectable label,and then examining the location and quantity of the label. The label maybe a known label, such as radioactive label, e.g. Tecnecium-99, andfluorescent label. Methods for labeling cells are also known. Detectionof labels and quantification of signals are also known in the art; theycan be performed using a radiation counter, by fluorescence assay, or byobtaining a tissue sample by biopsy.

The present invention is illustrated in greater detail below withreference to Examples, but it should be understood that the presentinvention is not construed as being limited thereto.

EXAMPLE 1

A. Experimental Methods and Materials Used

(1) Cells

Peripheral blood samples were obtained from five healthy volunteers (HV1to HV5) and four HLA-A*2402-positive AML patients (AML1 to AML4). Bonemarrow samples and peripheral blood samples were obtained from fiveHLA-A*2402-positive MDS patients (MDS1 to MDS5). More specifically,peripheral blood was obtained before treatment and at weeks 4, 8, 12,and 42 of treatment from ALM1. Peripheral blood was obtained beforetreatment and at weeks 4, 8, 12, and 42 of treatment from ALM2.Peripheral blood was obtained before treatment and at weeks 4 and 8 oftreatment from ALMS. Peripheral blood was obtained before treatment andat week of treatment from ALM4. From MDS1, bone marrow and peripheralblood were obtained before treatment; and peripheral blood was obtainedat weeks 4, 8, and 12 of treatment. Peripheral blood and bone marrowbefore treatment were collected from MDS1 to MDS5. The peripheral bloodsamples obtained were subjected to Ficoll-Hypaque (Pharmacia, Uppsala,Sweden) density gradient centrifugation and peripheral blood mononuclearcells (PBMCs) were separated and stored frozen at −170° C. until use.The treatment on the patients was conducted using WT1 peptideimmunotherapy. The amino acid sequence of the WT1 peptide used in thetreatment is Cys-Tyr-Thr-Trp-Asn-Gln-Met-Asn-Leu (SEQ ID No.: 3393).This peptide was administered by intradermal injection at an interval oftwo weeks.

(2) Flow cytometric analysis and sorting

Initially, 2×10⁶ PBMCs per sample were stained with PE-conjugatedHLA-A*2402-WT1 235-243 tetramers (MBL, Tokyo, Japan) in FACS buffer (PBScontaining 2% fetal bovine serum) for 30 minutes at 37° C. Subsequently,they were stained with monoclonal antibodies labeled with five differentfluorescent dyes as described below for 25 minutes on ice in dark:FITC-labeled anti-CD4, CD14, CD16, CD19, and CD56, anti-CD3-PerCP,anti-CD8-APC-Cy7 (BD Bioscience, San Jose, Calif.), anti-CD45RA-APC, andanti-CCR7-PE-Cy7 (BD Pharmingen, San Diego, Calif.). The stained cellswere washed twice in FACS buffer. Sorting was performed using theFACSAria system (BD Biosciences) and data analysis was performed usingthe FACSDiva software (BD Biosciences). As a result, singleHLA-A*2402-WT1 235-243 tetramer⁺CD3⁺CD8⁺ cells were obtained from thefraction of CD4⁻CD14⁻CD16⁻CD19⁻CD56⁻ cells and were defined as WT1-Tet⁺cells.

(3) cDNA Synthesis of the TCR-β Chain from the Single Cells Sorted

To eliminate DNA contamination for RT-PCR of sufficient single cells,cDNA synthesis was carried out, and all the steps of PCR were performedin a different clean bench. Single WT1-Tet⁺ cells were sorted directlyin a PCR tube containing 15 μl of a cDNA reaction mixture. The cDNAreaction mixture contained the following components in lysis buffer (1×cDNA buffer containing 0.5% Triton X-100): reverse transcriptase(SuperScript III, Invitrogen, Carlsbad, Calif.), 0.5 mM dNTPs(Invitrogen), 20 units (U) of Rnase inhibitor(Invitrogen), 100 μg/mlgelatin (Roche, Indianapolis, Ind.), 100 pg/ml tRNA (Roche), and a 200nM primer (5′-CACCAGTGTGGCCTTTTG-3′ (SEQ ID No.: 3394)) specific for theTCR-β chain constant region. After the sorting, the samples wereincubated for 90 minutes at 50° C. for cDNA synthesis, and thenincubated for 5 minutes at 95° C. for terminating the reaction.

(4) Semi-Nested Multiplex-PCR

Ten μl of a synthesized cDNA product obtained by the above procedure wasadded to 40 μl of a reaction mixture. The reaction mixture contained 1>PCR buffer, 2 mM MgCl₂, 0.25 mM dNTPs, 1.25 U DNA polymerase (PlatinumTag DNA Polymerase, Invitrogen), a 5 nM mixture of 24 differentVβ-family-specific forward primers (shown in Table 1), and 5 nM 3′-Cβreverse primer (5′-GCTTCTGATGGCTCAAACACAGC-3′ (SEQ ID No.: 3395)). Theprocedure of PCR was as follows: a pre-PCR heating step at 95° C. for 2minutes, followed by 40 cycles of a denaturing step at 95° C. for 45seconds, an annealing step at 57° C. for 45 seconds, and an extensionstep at 72° C. for 50 seconds.

TABLE 1 S2 TRBV2 GGTCACACAGATGGGACAGGAAGT (SEQ ID No.: 3396) S2 TRBV3CCCAGACTCCAAAATACCTGGTCA (SEQ ID No.: 3397) S5 TRBV4TACGCAGACACCAA<GA>ACACCTGGTCA (SEQ ID No.: 3398) S1 TRBV5ACAGCAAGTGAC<TAG>CTGAGATGCTC (SEQ ID No.: 3399) S7 TRBV6GTGTCACTCAGACCCCAAAATTCC (SEQ ID No.: 3400) S4 TRBV7-1GTGTGATCCAATTTCAGGTCATAC (SEQ ID No.: 3401) S3 TRBV7-3ATGTAACT<CT>TCAGGTGTGATCCAA (SEQ ID No.: 3402) S1 TRBV9ACAGCAAGTGAC<TAG>CTGAGATGCTC (SEQ ID No.: 3403) S1 TRBV10CCAAGACACAAGGTCACAGAGACA (SEQ ID No.: 3404) S4 TRBV11CAGTCTCCCAGATATAAGATTATAGAG (SEQ ID No.: 3405) S4 TRBV12GGTGACAGAGATGGGACAAGAAGT (SEQ ID No.: 3406) S8 TRBV13CTGATCAAAGAAAAGAGGGAAACAGCC (SEQ ID No.: 3407) S6 TRBV14ATAGAAGCTGGAGTTACTCAGTTC (SEQ ID No.: 3408) S8 TRBV15CAAGATACCAGGTTACCCAGTTTG (SEQ ID No.: 3409) S5 TRBV18TGCAGAACCCAAGACACCTGGTCA (SEQ ID No.: 3410) S5 TRBV19CACTCAGTCCCCAAAGTACCTGTT (SEQ ID No.: 3411) S2 TRBV20GAGTGCCGTTCCCTGGACTTTCAG (SEQ ID No.: 3412) S6 TRBV21AAGGTCACCCAGAGACCTAGACTT (SEQ ID No.: 3413) S6 TRBV23ACAAAGATGGATTGTACCCCCGAA (SEQ ID No.: 3414) S7 TRBV24GTTACCCAGACCCCAAGGAATAGG (SEQ ID No.: 3415) S1 TRBV25GATCACTCTGGAATGTTCTCAAACC (SEQ ID No.: 3416) S3 TRBV27GTGACCCAGAACCCAAGATACCTC (SEQ ID No.: 3417) S2 TRBV28GTAACCCAGAGCTCGAGATATCTA (SEQ ID No.: 3418) S3 TRBV29TCCAGTGTCAAGTCCATAGCCAAGTC (SEQ ID No.: 3419) S5 TRBV30GTCAGATCTCAGACTATTCATCAATGG (SEQ ID No.: 3420)

Next, the PCR products were subjected to screening PCR. One μl of eachPCR product was placed in separate 8 tubes and 24 μl of a reactionmixture was added to each of the tubes. The reaction mixture contained1× PCR buffer, 2 mM MgCl₂. 0.2 mM dNTPs, 1.0 U of Platinum Tag DNApolymerase, one set of forward primers selected from the 8 screeningsets S1 to S8, shown in Table 1, and 5′-Cβ reverse primer(5′-GGAACACGTTTTTCAGGTCCT-3′ (SEQ ID No.: 3421)) (150 nM each). Theprocedure of PCR was as follows: a pre-PCR heating step at 95° C. for 2minutes, followed by 35 cycles of a denaturing step at 94° C. for 45seconds, an annealing step at 57° C. for 45 seconds, and an extensionstep at 72° C. for 40 seconds.

To verify positive reactions in the 8 screening PCRs, 5 μl of eachscreening PCR product was subjected to 2% agarose gel electrophoresis,followed by further PCR. This PCR was performed 35 cycles using 150 nMeach of the VP-specific forward primers contained in the screening setsthat were confirmed as positive. The positive reaction was verified by2% agarose gel electrophoresis. A cell-free system was used as negativecontrast, and three wells were prepared for the negative contrast in thesame manner as above. The experiment was performed according to the sameprocedure.

(5) Determination and Analysis of the Sequences of theComplementarity-Determining region 3 (CDR3) of TCR-β

Amplified fragments of the TCR-β gene were purified using the PCRPurification Kit (Qiagen, Valencia, Calif.). Corresponding TCRVR-specific forward primers were used for sequencing. The ABI PRIAMBigDye Terminator v 3.1 Cycle Sequencing kit (Applied Biosystems, FosterCity, Calif., USA) was used for sequencing, and the ABI PRISM 3100Genetic Analyzer (Applied Biosystems) was used for analysis. Thesequence data on CDR3 were analyzed by comparing the sequences withthose available from the website of the IMGT human TCR gene database(imgt.cines.fr).

B. Results

The sequences of the gene for the Vβ chain, the J region sequences, Dregion sequences, N region sequences, CDR3 nucleotide sequences, andCDR3 amino acid sequences of WT1-specific CTLs derived from healthyindividuals and cancer patients (AML1 to AML4 and MDS1 to MDSS) areshown in FIGS. 1A-1 to 1ZZZ-3. The clonality of each WT1-specific CTL isshown in FIGS. 2A-1 to 2VVVV-3. The CDR3 nucleotide sequences are shownin SEQ ID Nos.: 1-1696 (since clones #24.29 and #28.92 have theidentical CDR3 nucleotide sequence, only clone #24.29 is shown in theSequence Listing), and the CDR3 amino acid sequences are shown in SEQ IDNos.: 1697-3392 (since clones #2.53 and #28.58 have the identical CDR3amino acid sequence, and so do clones #24.29 and #28.92, only clones#2.53 and #24.19 are shown in the Sequence Listing). Among the AMLpatients, AML1 and AML2 responded to the treatment, but AML3 and AML4did not. The response of the MDS patients to the treatment has not beendetermined.

As can be seen from FIGS. 2A-1 to 2VVVV-3, in the healthy individuals(HV1 to HV4), the clonality of WT1-specific CTL having any of the CDR3polynucleotides or CDR3 peptides is 1 for almost all the clones, and 2or 3 for rare clones; however, all the cancer patients before therapy(AMLs and MDSs) have a multiplicity of clonality of the WT1-specificCTLs having any of the CDR3 polynucleotides or CDR3 peptides withoutexception. The number of the clonality was larger and the types of suchcells were more abundant in the cancer patients than in the healthyindividuals.

The increases in clonality and in types of cells having a multiplicityof clonality in patients before therapy indicate a possibility thatdefense and attack against cancer cells has already been launched in thepatients and suggest that cancer cell are already present in thepatients.

Also, as can be seen from FIGS. 2A-1 to 2VVVV-3, the AML patients whoresponded to WT1 peptide immunotherapy had a larger number of types ofclones with a multiplicity of clonality than the healthy subjects andnon-responsive patients before the therapy. AML1 (responder): 10 types(clonality of 58 in total); AML2 (responder): 12 types (clonality of 93in total); AML3 (non-responder): 9 types (clonality of 27 in total); andAML4 (non-responder): 3 types (clonality of 10 in total). Healthyindividuals HV1-5 had clones with a multiplicity of clonality in a rangeof 0 to 2 types.

The clones that already have a multiplicity of clonality before thetherapy tend to further increase the clonality or maintain theirclonality by WT1 peptide immunotherapy. It is also considered that WT1peptide immunotherapy is more likely to be successful when there areclones of as many types as possible, which gives increased number andtypes of effective WT1-specific CTL clones as a whole.

As can be seen from FIGS. 2A-1 to 2VVVV-3, WT1-specific CTLs wereobserved in which the clonality increased either temporarily orcontinuously (including the cases where the clonality increased from 0to 1 or more) in the AML patients during the period of WT1 peptideimmunotherapy. The exemplary clones exhibiting such behavior includeclones #02.28, #05.034, #05.135, #05.219, #09.65, #12.04, #13.13,#27.016, and #28.83 from AML (patient 1—a responder), and clones #02.27,#05.035, #05.141, #05.219, #06.009, #12.04, #12.20, #15.44, #20.068, and#27.032 from AML (patient 1—a non-responder). For example, from thechange in the clonality of clone #02.28 from AML patient 1, it is foundthat the therapeutic effect was high at the onset of the treatment andat week 4.

An increase in the clonality of a certain clone indicates that the WT1peptide immunotherapy showed its effect for a certain period of time.Even if the clonality of certain clones may increase temporarily andthen decrease, the clonality of other WT1-specific CTLs would increaseto complement the effect. Considering the effect on cancer cells, alarger increase in the clonality is more desired. Therefore, the largerbecomes the increase in the clonality or the more abundant become thetypes of clones with increased clonality after the WT1 peptideimmunotherapy, the responsiveness to the WT1 peptide immunotherapy isconsidered to have been high.

INDUSTRIAL APPLICABILITY

The present invention may provide pharmaceutical compositions useful foranti-cancer therapy, cancer test kits or reagents, reagents for cancerresearch, and the like. Therefore, the present invention may find use inthe fields of pharmaceuticals for cancer therapy, of cancer test kits orreagents, and of cancer research.

1. A method for assaying the clonality of a WT1-specific CTL,comprising: (a) sorting single WT1-specific CTLs in a sample; (b)contacting a peptide from a single WT1-specific CTL with an antibodyagainst a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1697-3392; and (c) measuring theclonality of WT1-specific CTLs that comprise an identical amino acidsequence selected from the group consisting of SEQ ID NOs: 1697-3392,thereby characterizing said WT1-specific CTLs as having a multiplicityof clonality if said clonality is greater than 1; and/or measuring thenumber of types of WT1-specific CTLs that have a multiplicity ofclonality.
 2. The method of claim 1, wherein the antibody in step (b) isimmobilized on a chip.
 3. A method for diagnosing cancer in anHLA-A*2402-positive subject, comprising assaying the clonality of aWT1-specific CTL using the method of claim 1 in a sample obtained fromthe subject before a therapy, wherein the subject is determined to havean increased possibility of developing cancer when a WT1-specific CTLhaving a multiplicity of clonality is present.
 4. The method of claim 3,wherein an increased clonality of WT1-specific CTLs having a clonalityof 3 or more, or an increasingly abundant number of types ofWT1-specific CTLs having a clonality of 3 or more, indicates anincreased possibility of developing cancer.
 5. A method for testing thesensitivity of an HLA-A*2402-positive subject to WT1 peptideimmunotherapy, comprising assaying the clonality of a WT1-specific CTLusing the method of claim 1 in a first sample obtained from the subjectbefore the immunotherapy and in a second sample obtained from a subjectnon-responsive to the immunotherapy, wherein the subject is determinedto have sensitivity to the immunotherapy when the types of WT1-specificCTLs having a multiplicity of clonality in the subject are more abundantthan in the subject non-responsive to the immunotherapy.
 6. The methodof claim 5, wherein an increasingly abundant number of types ofWT1-specific CTLs having a multiplicity of clonality indicates anincreased sensitivity to the immunotherapy.
 7. A method for monitoringWT1 peptide immunotherapy in an HLA-A*2402-positive subject, comprisingassaying the clonality of a WT1-specific CTL using the method of claim 1in a first sample obtained from the subject before the immunotherapy andin a second sample obtained from the subject after the immunotherapy,wherein the subject is determined to have responded to the immunotherapywhen the clonality of a WT1-specific CTL increases after theimmunotherapy compared to before the immunotherapy.
 8. The method ofclaim 7, wherein an increased clonality of WT1-specific CTLs after theimmunotherapy, or an increasingly abundant number of types ofWT1-specific CTLs having a multiplicity of clonality after theimmunotherapy, indicates increased responsiveness to the immunotherapy.